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. 2013 Apr;16(2):373-86.
doi: 10.1007/s10456-012-9321-x. Epub 2012 Nov 10.

Concentration- And Schedule-Dependent Effects of Chemotherapy on the Angiogenic Potential and Drug Sensitivity of Vascular Endothelial Cells

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Free PMC article

Concentration- And Schedule-Dependent Effects of Chemotherapy on the Angiogenic Potential and Drug Sensitivity of Vascular Endothelial Cells

Eddy Pasquier et al. Angiogenesis. .
Free PMC article

Abstract

The anti-angiogenic activity of chemotherapy is both dose- and schedule-dependent. While conventional maximum tolerated dose (MTD) chemotherapy exerts only mild and reversible anti-angiogenic effects, low-dose metronomic (LDM) chemotherapy was developed to specifically target tumour angiogenesis. However, the long-term effects of either MTD or LDM chemotherapy on vascular endothelial cells have never been investigated. Here, we demonstrated that repeated exposure to MTD and LDM chemotherapy differentially impact on the angiogenic potential and chemosensitivity of immortalized endothelial cells. Repeated MTD vinblastine treatment of vascular endothelial cells led to an increased proliferation rate and resistance to paclitaxel. In contrast, repeated LDM treatment with vinblastine or etoposide impaired the angiogenic potential of endothelial cells and increased their chemosensitivity. This effect was associated with a significant decrease in βII- and βIII-tubulin expression. Functional analysis using siRNA showed that silencing the expression of βIII-tubulin in endothelial cells significantly decreased their capacity to form vascular structures and increased their sensitivity to the anti-angiogenic and vascular-disrupting effects of chemotherapy, whereas silencing βII-tubulin expression had no effect. Collectively our results show that LDM chemotherapy impairs the angiogenic potential of endothelial cells while increasing their chemosensitivity-an effect at least in part mediated by the down-regulation of βIII-tubulin expression. Furthermore, our study suggests that βIII-tubulin represents an attractive therapeutic target to increase the anti-angiogenic effects of chemotherapy and overall anti-tumour efficacy.

Figures

Fig. 1
Fig. 1
Impact of repeated exposure to chemotherapy on the angiogenic potential of endothelial cells. a Doubling time of the 6 endothelial subclones established by incubating BMH29L endothelial cells for 100 days with no drug (Ctrl-LDM and Ctrl-MTD) or with vinblastine (VLB) or etoposide (VP16) following a low-dose metronomic (LDM) schedule (i.e. treatment 5 days/week with highest non-toxic concentration) or a maximum tolerated dose (MTD) schedule (i.e. treatment every 2 weeks with IC80). Columns means of four individual experiments, bars SE. Statistics were calculated by comparing drug-treated cells with control untreated cells, unless indicated otherwise; *p < 0.05; **p < 0.01. b Representative photographs of the 6 BMH29L subclones following 8 h incubation on Matrigel. Vascular structures were imaged on a Zeiss Axiovert 200 M using a 5X objective. Percentage of angiogenesis inhibition as compared to control cells is indicated; NS non-significant, Scale bar 250 μm
Fig. 2
Fig. 2
Impact of repeated exposure to chemotherapy on the chemosensitivity of endothelial cells. ac Histograms showing the IC50 values of vinblastine (a), etoposide (b) and paclitaxel (c) in the 6 BMH29L subclones as determined by 72 h growth inhibition assay. df Percentage of vascular structure formation following 8 h incubation on Matrigel with 5 nM vinblastine (d), 10 μM etoposide (e) and 10 nM paclitaxel (f). Columns means of at least four individual experiments, bars SE. Statistics were calculated by comparing drug-treated cells with control cells, unless indicated otherwise; *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3
Fig. 3
Impact of repeated exposure to chemotherapy on ABC transporter expression and intracellular drug accumulation. a Relative gene expression of ABC transporters as determined by qRT-PCR using HPRT1 as control gene. b Accumulation of [3H]-VCR after 4 h incubation in presence or absence of 10 μM verapamil. The BE(2)-C neuroblastoma cell line expressing high levels of P-gp was included as a positive control. Columns means of four individual experiments, bars SE. Statistics were calculated by comparing drug-treated cells with control cells, unless indicated otherwise; #p = 0.06; *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 4
Fig. 4
Effect of repeated exposure to chemotherapy on the expression of β-tubulin isotypes in endothelial cells. a Representative immunoblots of total endothelial cell lysates following long-term treatment with chemotherapy. Membranes were probed with antibodies directed against GADPH (loading control), βI-, βII-, βIII-, βIV- and total β-tubulin. b Histogram showing the relative expression of tubulin isotypes in the 6 BMH29L subclones as determined by densitometry after normalization with GADPH. Columns means of three individual experiments, bars SE. Statistics were calculated by comparing drug-treated cells with control cells; *p < 0.05; **p < 0.01
Fig. 5
Fig. 5
Functional study of βII- and βIII-tubulin in endothelial cells. a Representative immunoblots of whole cell lysates, 72 h after transfection of HMEC-1 cells with negative control, βII- and βIII-tubulin siRNA. Membranes were probed with antibodies directed against GADPH (loading control), βI-, βII-, βIII- and βIV-tubulin. b Mean surface occupied by vascular structures formed by HMEC-1 cells 72 h after siRNA transfection and following 8 h incubation on Matrigel. Boxes min–max range of at least 6 individual experiments, bars SD; ***p < 0.001. cf Growth inhibition assays performed on siRNA-transfected HMEC-1 cells using Alamar Blue after 72 h incubation with a range of concentrations of vinblastine (c), etoposide (d), paclitaxel (e) and 2-methoxyestradiol (f). Points % of cell proliferation as compared to untreated control cells, means of at least three individual experiments, bars SE; log scale for x axis
Fig. 6
Fig. 6
Impact of βII- and βIII-tubulin knockdown on the sensitivity of endothelial cells to the anti-angiogenic effects of chemotherapy. a Representative photographs of siRNA-transfected HMEC-1 cells incubated for 8 h on Matrigel in the absence of drug (top panel) or in presence of vinblastine at 2 nM (middle panel) and etoposide at 5 μM (bottom panel). Vascular structures were imaged on a Zeiss Axiovert 200 M using a 5X objective. Percentage of angiogenesis inhibition as compared to untreated control siRNA-transfected cells is indicated; NS non-significant, scale bar 250 μm. b Percentage of vascular structure formation by siRNA-transfected HMEC-1 cells following 8 h incubation on Matrigel with 2 nM vinblastine (VLB), 2 μM 2-methoxyestradiol (2ME2), 5 nM paclitaxel (PTX), 5 μM etoposide (VP16) and 10 μM mafosfamide (MFA), as compared to control untreated cells. Columns means of at least three individual experiments, bars SE. Statistics were calculated by comparing the mean surface occupied by closed vascular structures per view field (at least 10 view fields per condition); *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 7
Fig. 7
Impact of βII- and βIII-tubulin knockdown on the sensitivity of endothelial cells to vascular-disrupting agent ENMD-1198. a Representative photographs of siRNA-transfected HMEC-1 cells in vascular-disruption assay. Cells were first allowed to form vascular structures on Matrigel for 6 h before drug treatment was initiated. Cells were then incubated for 2 h in presence of ENMD-1198 at 0.25 μM and vascular structures were imaged on a Zeiss Axiovert 200 M using a 5X objective. Arrows point to collapsing and regressing vascular structures. Percentage of vascular disruption is indicated; Scale bar 250 μm. b Dose-dependent effect of ENMD-1198 on the disruption of capillary-like structures formed by siRNA-transfected HMEC-1 cells, after 2 h drug incubation. Points % of intact vascular structures as compared to untreated control cells, means of at least three individual experiments, bars SE; log scale for x axis. Statistics were calculated by comparing the mean surface occupied by closed vascular structures per view field (at least 10 view fields per condition); *p < 0.05; ***p < 0.001

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